Today, the most important protective measure against a viral infection and for limiting its spread is preventive vaccination. Modern vaccines, as a rule, induce the formation of antibodies to surface viral antigens. Vaccine effectiveness directly depends on the degree of matching between the antigenic structure of the virus strains containing in a vaccine and the strains circulating in the population. Surface proteins of the majority of viruses undergo constant antigenic variation (antigenic drift), necessitating constant updating of vaccine strain composition. The development of highly immunogenic and safe vaccines inducing the immune response of a broad spectrum of action is currently one of the major problems encountered in efficient influenza prevention.
Of all the viral respiratory diseases, influenza causes the most severe pathology and leads to the greatest damage to the population health and economy. The lack of population immunity to the periodically emerging new pandemic influenza strains makes influenza infection especially dangerous. It is known that the Spanish flu caused the death of 30 to 50 million people in 1918. Currently, according to the World Health Organization (WHO) data, each year approximately 20% of the population worldwide, including 5-10% of adults and 20-30% of children, become ill with influenza during seasonal epidemics (World Health Organization) at the website www.who.int under the directory biologicals/vaccines/influenza/en/ (accessed date: 28.03.2016)). Severe disease forms are recorded for 3-5 million cases, and 250,000 to 500,000 cases are lethal. Economic losses caused by influenza and other acute respiratory viral infections (ARVI) account for approximately 77% of the total damage from all infectious diseases. Significant losses are related both to the direct costs of patients' treatment and rehabilitation, as well as to the indirect losses caused by a decrease in productivity and reduction in corporate profits. Influenza and acute respiratory viral infections account for 12-14% of the total number of temporary disability cases.
The existing vaccines can be subdivided into two types: the attenuated (live, containing whole and active viruses exhibiting low pathogenicity) and inactivated (containing fragments of viral particles or whole inactive viruses) types. Live viruses that can replicate in an infected host elicit a strong and long-lasting immune response against the expressed antigens of these viruses. They effectively induce both humoral and cellular immune responses, and stimulate cytokine- and chemokine-mediated immune responses. Therefore, live attenuated viruses have certain advantages over vaccine compositions based on either inactivated immunogens or separate subunits of an immunogen, which generally stimulate only the humoral part of the immune system.
For vaccination of animals and humans from various infectious diseases, viruses of different families can be used as vectors expressing foreign genomic sequences. Vectors can be used in the cases where traditional killed or live vaccines cannot be produced or their effectiveness does not allow control of a disease. Among the existing antigen delivery systems, viral vectors occupy a special place because of the following properties: they have a natural mechanism of interaction with a cell and penetration into it, transfer foreign genetic material to the cytoplasm or nucleus of a cell, and are able to provide long-lasting expression of an antigen, and the viral envelope protects the nucleic acid encoding an introduced transgene.
Not all viruses have the properties necessary to construct vectors for the production of effective attenuated recombinant vaccines. Currently, for the development of viral vector-based vaccines, most widely used viruses are poxviruses (Poxviridae) [J. Gen. Virol. 2005. V. 86. No. 11. P. 2925-2936], Newcastle disease virus (NDV) [Virol. 2001. V. 75. No. 23. P. 11868-11873] and adenoviruses (Adenoviridae) [Biotechnology. 2007. V. 5, P. 38-44]. Among the poxviruses used as a viral vector, the most popular virus is vaccinia virus having advantages, such as simplicity and low cost of production, as well as a high packing capacity (up to 25 kbp) [J. Gen. Virol. 2005. V. 86. No. 11. P. 2925-2936]. A serious disadvantage of vaccinia virus-based vectors is pre-existing immunity to this virus, which is present in a part of the human population as a result of immunization against smallpox. Therefore, it is advisable to use vectors based on poxviruses, such as canarypox (Canarypox) and poultry poxvirus (Flowpox). However, Canarypox and Flowpox induce weaker immune response to target antigens than the vaccinia virus and require repeated administration or use of adjuvants [Vaccine. 1991. V. 9. No. 5, P. 303-308]. A significant disadvantage of a NDV vaccine vector is that the effects of the administration of recombinant NDVs have not been sufficiently studied, and it is not clear whether NDV-based vaccines are safe for humans. In addition, NDV is characterized by a low packing capacity and difficulty in producing vectors carrying several target antigens [Chem. Biodivers. 2010. V. 7. No. 3. P. 677-689]. Adenoviruses also have a number of disadvantages limiting their use as vectors for gene transfer. The major disadvantages of adenoviral vectors are the following: (1) heterogeneous distribution of the viral receptors on the surface of cells in the body, which makes many cells insensitive to adenovirus infection; (2) the presence of a powerful protective immunity of the population to known adenoviral vectors; and (3) a theoretical possibility of integration of the adenovirus DNA genome into human chromosomes (Stephen S L, Montini E, Sivanandam V G, Al-Dhalimy M, Kestler H A, Finegold M, Grompe M, Kochanek S. Chromosomal integration of adenoviral vector DNA in vivo. J Virol. 2010 October; 84(19):9987-94. doi: 10.1128/JVI.00751-10. Epub 2010 Aug. 4).
Vectors constructed based on influenza virus have several advantages over other viral vectors, because:                influenza viruses do not have a DNA phase in their replication cycle and cannot be inserted into the human or animal genome;        influenza virus elicits systemic and mucosal B- and T-cell responses to its antigens upon infection of human respiratory tract cells;        there are available multiple different influenza virus subtypes. Since antibodies to said various subtypes do not have cross-reactivity, it is possible to avoid pre-existing immunity to a viral vector in a host, which is often a problem with other live vectors. Effective booster immunizations are also possible with various influenza virus subtypes that express the same antigens;        there are several types of live influenza vaccines for intranasal administration (LIVE allantoic INFLUENZA VACCINE ULTRAVAC® (RF) and Flumist® (USA)) and industrial technology of their production by using 10-day-old chicken embryos (Guideline on Influenza Vaccines—Quality Module, European Medicines Acency, 25 Apr. 2014 [electronic resource] at the website www.ema.europa.eu under the directory docs/en_GB/document library/Scientific_guideline/2014/06/WC500167817.pdf (accessed date: 11, Jan. 2015)).        
The influenza virus belongs to the family of Orthomyxoviridae, which includes genera: influenza A, B, and C viruses. Genomes of influenza A and B viruses are structurally similar, and consist of eight RNA genome segments of negative polarity: PB2, PB1, PA, HA, NA, NP, M and NS (Chou Y Y, Vafabakhsh R, Doğanay S, Gao Q, Ha T, Palese P. One influenza virus particle packages eight unique viral RNAs as shown by FISH analysis. Proc Natl Acad Sci USA. 2012 Jun. 5; 109(23):9101-6. doi: 10.1073/pnas.1206069109. Epub 2012 Apr. 30). The polymerase complex PB2, PB1, and PA transcribes one mRNA from each genomic fragment, which is translated to the protein of the same name. Messenger RNAs of genomic segments M and NS may be alternatively spliced to form mRNAs encoding M2 and NEP proteins, respectively. All proteins except NS1 and PB1-F2 (are available not in all strains) are structural components of a virus particle. Nonstructural protein NS1 accumulates in the cytoplasm of infected cells and acts as an interferon inhibitor (Krug R M. Functions of the influenza A virus NS1 protein in antiviral defense. Curr Opin Virol. 2015 June; 12:1-6. doi: 10.1016/j.coviro.2015.01.007. Epub 2015 Jan. 29. Review).
The segmented structure of the influenza virus genome is an source of new different strains that are the result of the reassortment process. This is one of the mechanisms for the natural antigenic diversity of influenza viruses and the occurrence of influenza pandemics.
The antigenic properties of influenza virus are determined by the surface glycoproteins—hemagglutinin (HA) and neuraminidase (NA) that form spikes on the virion surface. The HA molecule is responsible for the mechanisms of binding the virus to sialic acid receptors on a cell and fusing the viral and cell membranes for penetration of the genome into the cytoplasm and nucleus of the cell. In the process of viral replication, the HA is cleaved (HA activation) by cellular proteases into two subunits—HA1 and HA2—that remain connected by a disulfide bond (Bullough P A, Hughson F M, Skehel J J, Wiley DC. Structure of influenza haemagglutinin at the pH of membrane fusion. Nature 1994; 371, 37-43). The HA molecule consists of two parts: a globular part comprising HA1 subunit and the stem region, which is formed mainly by HA2 and partially by the HA1 subunit. The globular part includes a receptor-binding site and five antigenic sites, and serves as the main target for the formation of antibodies. Antibodies that block virus binding to the cell receptor are neutralizing. The HA1 subunit is characterized by high variability. The HA stem that is located in close proximity to the viral membrane is highly conservative and characterized by low immunogenicity. The main function of the HA2 subunit is to ensure the fusion of the viral and the endosomal membranes; this subunit is highly conserved. According to the antigenic specificity, 18 subtypes of HA and 11 subtypes of NA are known to date for the influenza A virus. The subtypes H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, and H18 belong to the first group, and the subtypes H3, H4, H7, H10, H14, and H15 belong to the second group. At the same time, only the subtypes H1, H2, and H3 of influenza virus A and different antigenic variants of influenza virus B, which are circulating in the human population, are causing the pandemics and seasonal influenza epidemics.
The specific immunity generated after the disease or after vaccination by one influenza A virus subtype poorly protects from infection by the other virus subtypes. The immunity to any influenza virus A subtype does not protect from the infection by influenza virus B, and vice versa—immunization against the influenza virus B is not effective in regard to influenza virus A. In this regard, there is an urgent need for the development of a universal influenza vaccine effective against all known antigenic varieties of influenza A and B viruses.
Two mechanisms enable the extremely high variability of the influenza virus and, therefore, its ability to escape from the neutralizing antibodies: 1) accumulation of the point mutations leading to the change in the antigenic structure of the surface glycoproteins (antigenic drift) and 2) reassortment of the genomic segments. They lead to the emergence of new subtypes of viruses (antigenic shift) that can cause pandemics.
All of the existing influenza vaccines have low efficiency in elderly and infants (Jefferson T, Rivetti A, Di Pietrantonj C, Demicheli V, Ferroni E. Vaccines for preventing influenza in healthy children. Cochrane Database Syst Rev 2012; 8, CD004879; Osterholm M T, Kelley N S, Sommer A, Belongia E A. Efficacy and effectiveness of influenza vaccines: a systematic review and meta-analysis. Lancet Infect Dis 2011; 12, 36-44; Pfleiderer M, Trouvin J H, Brasseur D, Granstrom M, Shivji R, Mura M, Cavaleri M. Summary of knowledge gaps related to quality and efficacy of current influenza vaccines. Vaccine 2014; 32, 4586-91). Furthermore, these vaccines can protect against the circulating virus only if the vaccine virus has the same antigenic properties as the epidemic strain. It is the high variability of the surface antigens of influenza virus—HA and NA—that necessitates annual vaccination and the updating of vaccine composition. It should be mentioned that seasonal vaccines that are developed according to the WHO recommendations are not effective in the case of the occurrence of a new influenza pandemic virus strain that is fundamentally different from all of the circulating strains, as it happened in 2009 when the pandemic virus A/California/7/2009 (H1N1pdm09) emerged. One more example could be the low efficiency of the H3N2 component of the seasonal vaccine 2014 due to the emergence of the new antigenic variant of this virus subtype as a result of antigenic drift (Skowronski D M, Chambers C, Sabaiduc S, De Serres G, Dickinson J A, Winter A L, Drews S J, Fonseca K, Charest H, Gubbay J B, Petric M, Krajden M, Kwindt T L, Martineau C, Eshaghi A, Bastien N, Li Y. Interim estimates of 2014/15 vaccine effectiveness against influenza A(H3N2) from Canada's Sentinel Physician Surveillance Network, January 2015. Euro Surveill 2015; 20). During the last 60 years, a lot of vaccines were developed that have certain advantages and shortcomings; however, none of the existing vaccines can solve the problem of influenza morbidity control because of their incapability of inducing cross-protective immunity to constantly evolving influenza viruses. In this regard, there is an urgent need for the development of an effective universal influenza vaccine that provides a long-lasting broad cross-protective immunity and is able to protect against the influenza A and B viruses of all known subtypes.
The function of all the known influenza vaccines inactivated (whole virion, split, or subunit) or live (attenuated cold adapted)—is to generate the immunity to the globular part of HA. In contrast to the variable globular part, the HA stem part of influenza A (groups I and II) and B viruses is much more conservative. There are known several mechanisms of direct and indirect neutralization for the antibodies induced to this part of HA. One of the mechanisms of direct neutralization contributes to the prevention of the HA conformational change that is necessary for the fusion peptide release and the subsequent fusion of the endosomal and viral membranes in order to deliver the viral genome into the cell. The second mechanism of the direct neutralization contributes to the prevention of HA cleavage to HA1 and HA2 subunits by antibodies interacting with the HA part that is located in the vicinity of the cleavage site. The antibody-dependent and complement-dependent cytotoxicity are involved in the mechanisms of indirect neutralization (Terajima M, Cruz J, Co M D, Lee J H, Kaur K, Wrammert J, Wilson P C, Ennis F A. Complement-dependent lysis of influenza a virus-infected cells by broadly cross-reactive human monoclonal antibodies. J Virol 2011; 85, 13463-7; Jegaskanda S, Weinfurter J T, Friedrich T C, Kent S J. Antibody-dependent cellular cytotoxicity is associated with control of pandemic H1N1 influenza virus infection of macaques. J Virol 2013; 87, 5512-22).
Vaccination practically does not induce the antibodies to the HA stem region, while after the natural infection a small quantity of these antibodies could be detected (Moody M A, Zhang R, Walter E B, Woods C W, Ginsburg G S, McClain M T, Denny T N, Chen X, Munshaw S, Marshall D J, Whitesides J F, Drinker M S, Amos J D, Gurley T C, Eudailey J A, Foulger A, DeRosa K R, Parks R, Meyerhoff R R, Yu J S, Kozink D M, Barefoot B E, Ramsburg E A, Khurana S, Golding H, Vandergrift N A, Alam S M, Tomaras G D, Kepler T B, Kelsoe G, Liao H X, Haynes B F. H3N2 influenza infection elicits more cross-reactive and less clonally expanded anti-hemagglutinin antibodies than influenza vaccination. PLoS ONE 2011; 6, e25797).
The majority of the currently being developed approaches to the generation of the universal vaccine are targeting the conservative regions of the influenza virus proteins. The antibodies directed to the conservative proteins PB2, PB1, PA, NP, and M1 do not have neutralizing activity but could play an important role in virus elimination by means of antibody-dependent cytotoxicity (ADCC).
Several examples of generating a universal vaccine are based on HA2 subunit. The triple immunization of mice with peptides representing the ectodomain HA2 (23-185 amino acid residues) or the fusion peptide (1-38 amino acid residues) conjugated to the (keyhole limpet hemocyanin) (KLH) and Freund adjuvants induced the cross-reactive immunity leading to a decrease in the animal mortality when challenged with a lethal dose of heterologous virus strain (Stanekova Z, Kiraly J, Stropkovska A, Mikuskova T, Mucha V, Kostolansky F, Vareckova E. Heterosubtypic protective immunity against influenza A virus induced by fusion peptide of the hemagglutinin in comparison to ectodomain of M2 protein. Acta Virol 2011; 55, 61-7). More effective protection was developed in the case of vaccination with chimeric HA constructs. Krammer et al. showed that heterosubtypic humoral immunity is induced in mice immunized with chimeric proteins, containing the HA globular parts from the viruses of different subtypes in combination with the HA stem region of the same virus (Krammer F, Palese P, Steel J. Advances in universal influenza virus vaccine design and antibody mediated therapies based on conserved regions of the hemagglutinin. Curr Top Microbiol Immunol 2014; 386, 301-21; Krammer F, Hai R, Yondola M, Tan G S, Leyva-Grado V H, Ryder A B, Miller M S, Rose J K, Palese P, Garcia-Sastre A, Albrecht R A. Assessment of influenza virus hemagglutinin stalk-based immunity in ferrets. J Virol 2014; 88, 3432-42). The complicated immunization scheme that includes the animals electroporation using DNA, and double intramuscular and intranasal immunization with the protein constructs supplemented with the adjuvant poly (I:C) are the shortcomings of this approach.
The use of stabilized structures (mini-HA) generated by means of gene engineering, based on the amino acid sequence of the HA stem region of the H1N1 virus, serves as an example of a different approach to the generation of the universal influenza vaccine. Only the structures with the highest affinity to the antibodies that have a broad range of neutralizing activity were selected from the large library. The immunization of mice with these structures also protected the animals from death when challenged with highly pathogenic avian influenza virus of H5N1 subtype (Impagliazzo A, Milder F, Kuipers H, Wagner M V, Zhu X, Hoffman R M, van Meersbergen R, Huizingh J, Wanningen P, Verspuij J, de Man M, Ding Z, Apetri A, Kukrer B, Sneekes-Vriese E, Tomkiewicz D, Laursen N S, Lee P S, Zakrzewska A, Dekking L, Tolboom J, Tettero L, van Meerten S, Yu W, Koudstaal W, Goudsmit J, Ward A B, Meijberg W, Wilson I A, Radosevic K. A stable trimeric influenza hemagglutinin stem as a broadly protective immunogen. Science 2015; 349, 1301-6). The complete protection of mice from death was achieved by the double intramuscular immunization with 30 μg of the purified mini-HA protein supplemented with the Matrix-M adjuvant produced by Novavax.
The other prospective direction in the development of the universal influenza vaccine is based on the design of the self-assembling nanoparticles that significantly enhance the immunogenic properties of HA (Kanekiyo M, Wei C J, Yassine H M, McTamney P M, Boyington J C, Whittle J R, Rao S S, Kong W P, Wang L, Nabel G J. Self-assembling influenza nanoparticle vaccines elicit broadly neutralizing H1N1 antibodies. Nature 2013; 499, 102-6). The animals were immunized 2 or 3 times intramuscularly with nanoparticles supplemented with the new adjuvant SAS (Sigma Adjuvant System). In spite of the lack of the neutralizing antibodies after immunization with nanoparticles, the mice as well as ferrets turned out to be completely protected from death when infected with a highly pathogenic H5N1 avian virus.
One of the modern technologies for the generation of live vaccine is based on the construction of vaccine vectors that enable to express the antigens of one virus by the other virus. Different DNA-containing viruses, namely: adenovirus, herpesvirus, baculovirus, or poxvirus, are used as the vectors for the expression of influenza antigens (Dudek T, Knipe D M. Replication-defective viruses as vaccines and vaccine vectors. Virology 2006; 344, 230-9; He F, Madhan S, Kwang J. Baculovirus vector as a delivery vehicle for influenza vaccines. Expert Rev Vaccines 2009; 8, 455-67; Draper S J, Cottingham M G, Gilbert S C. Utilizing poxviral vectored vaccines for antibody induction-progress and prospects. Vaccine 2013; 31, 4223-30. Price G E, Soboleski M R, Lo C Y, Misplon J A, Pappas C, Houser K V, Tumpey T M, Epstein S L. Vaccination focusing immunity on conserved antigens protects mice and ferrets against virulent H1N1 and H5N1 influenza A viruses. Vaccine 2009; 27, 6512-21). Thus, the experiments with the adenovirus vector showed that the triple immunization with plasmid (50 μg) containing the sequences of the influenza A virus conservative proteins NP and M2, followed by intranasal infection with the two adenovirus vectors that express the same proteins, led to the complete protection of the mice and ferrets infected with the virus A/FM/1/47 (H1N1) or with the highly pathogenic avian influenza virus H5N1 subtype, from death and weight loss.
Thus, all of the discussed approaches of targeting an immune response to the conservative regions of influenza virus antigens prove the possibility of the generation of a vaccine that will protect from infection with different variants of influenza A virus. However, complex schemes of multiple vaccinations of animals by using immunological adjuvants of different nature were used to achieve this goal. In addition, none of the known experimental preparations of a universal influenza vaccine provided protection against influenza B virus. It should be added to this that the above experimental preparations require complex technological processes for the production of multicomponent vaccines, associated with an unacceptably high cost of the final product.
Expression of antigens in cells of the nasal cavity is known to induce systemic and local mucosal B- and T-cell immune responses. Numerous attempts have been made to use influenza viruses as vectors for delivery and expression of foreign genomic sequences in cells of the respiratory tract of animals. Among 8 genomic fragments of influenza A or B viruses, only NS genomic fragment was capable of stably holding genomic insertions of more than 800 nucleotides in the reading frame of NS1 nonstructural protein, without disrupting the structure of the resulting virions (Kittel C, Sereinig S, Ferko B, Stasakova J, Romanova J, Wolkerstorfer A, Katinger H, Egorov A. Rescue of influenza virus expressing GFP from the NS1 reading frame. Virology. 2004 Jun. 20; 324(1):67-73. PubMed PMID: 15183054). Moreover, among all influenza virus proteins, only NS1 protein normally containing 230-237 amino acid residues can be truncated to 50% at the carboxyl end, without significantly affecting the reproductive activity of the virus in cell cultures, chicken embryos or in the respiratory tract of animals (Egorov A, Brandt S, Sereinig S, Romanova J, Ferko B, Katinger D, Grassauer A, Alexandrova G, Katinger H, Muster T. Transfectant influenza A viruses with long deletions in the NS1 protein grow efficiently in Vero cells. J Virol. 1998 August; 72(8):6437-41. PubMed PMID: 9658085; PubMed Central PMCID: PMC109801). This truncation of the NS1 protein provides a space for introduction of long insertions of foreign genomic sequences without disrupting the morphology and basic functions of the virus, thus making it possible to construct genetically stable vectors. In this regard, influenza virus vectors based on influenza A virus were produced that encoded a truncated reading frame of from 80 to 126 amino acid residues of the NS1 protein, wherein the truncated reading frame could be elongated by insertions of antigen sequences of various bacterial and viral pathogens, for example by the protein sequences of mycobacterium tuberculosis, brucella abortus or human immunodeficiency virus (Tabynov K, Sansyzbay A, Kydyrbayev Z, Yespembetov B, Ryskeldinova S, Zinina N, Assanzhanova N, Sultankulova K, Sandybayev N, Khairullin B, Kuznetsova I, Ferko B, Egorov A. Influenza viral vectors expressing the Brucella OMP16 or L7/L12 proteins as vaccines against B. abortus infection. Virol J. 2014 Apr. 10; 11:69. doi: 10.1186/1743-422X-11-69. PubMed PMID: 24716528; PubMed Central PMCID: PMC3997475; Sereinig S, Stukova M, Zabolotnyh N, Ferko B, Kittel C, Romanova J, Vinogradova T, Katinger H, Kiselev O, Egorov A. Influenza virus NS vectors expressing the mycobacterium tuberculosis ESAT-6 protein induce CD4+ Th1 immune response and protect animals against tuberculosis challenge. Clin Vaccine Immunol. 2006 August; 13(8):898-904. PubMed PMID: 16893990; PubMed Central PMCID: PMC1539114; Ferko B, Stasakova J, Sereinig S, Romanova J, Katinger D, Niebler B, Katinger H, Egorov A. Hyperattenuated recombinant influenza A virus nonstructural-protein-encoding vectors induce human immunodeficiency virus type 1 Nef-specific systemic and mucosal immune responses in mice. J Virol. 2001 October; 75(19):8899-908. PubMed PMID: 11533153; PubMed Central PMCID: PMC114458). The constructs carrying NS1 protein truncated to 124 amino acid residues (hereinafter, the NS1-124 vectors) appeared to be optimal by the parameters of reproduction in chicken embryos and of immunogenicity in animals (Ferko B, Stasakova J, Romanova J, Kittel C, Sereinig S, Katinger H, Egorov A. Immunogenicity and protection efficacy of replication-deficient influenza A viruses with altered NS1 genes. J Virol. 2004 December; 78(23):13037-45. PubMed PMID: 15542655; PubMed Central PMCID: PMC524997).
Constructs with a more truncated NS1 protein had a reduced ability to grow in interferon-competent cells (MDCK cells, A549), including a 10-day-old chicken embryos, and were suitable for the production only in interferon-deficient Vero cells. On the other hand, vectors with an NS1 protein consisting of 124-126 amino acid residues varied in attenuation and were not safe enough in animals. For example, the reproduction level of viral vectors carrying ESAT-6 mycobacterial protein at a specified position could reach in mouse lungs the values close to those of pathogenic influenza viruses (104 and more of virus particles per gram lung tissue). Moreover, NS1-124 vectors, at an infective dose of >5.0 log/mouse, could cause a significant reproduction of the virus in the lung tissue of infected mice and the formation of visible lung pathology (Egorov A, Brandt S, Sereinig S, Romanova J, Ferko B, Katinger D, Grassauer A, Alexandrova G, Katinger H, Muster T. Transfectant influenza A viruses with long deletions in the NS1 protein grow efficiently in Vero cells. J Virol. 1998 August; 72(8):6437-41. PubMed PMID: 9658085; PubMed Central PMCID: PMC109801; Stukova M A, Sereinig S, Zabolotnyh N V, Ferko B, Kittel C, Romanova J, Vinogradova T I, Katinger H, Kiselev O I, Egorov A. Vaccine potential of influenza vectors expressing Mycobacterium tuberculosis ESAT-6 protein. Tuberculosis (Edinb). 2006 May-July; 86(3-4):236-46. PubMed PMID: 16677861). Thus, influenza vectors with the NS1 reading frame truncated to 124 amino acid residues cannot be used for vaccination of humans because they do not correspond to the safety parameters developed for live influenza vaccines, where the essential condition is temperature sensitivity of the virus (a reduced reproductive ability at 39° C.) and the lack of active replication of the virus in the lower respiratory tract of animals (Maassab H F, Bryant M L. The development of live attenuated cold-adapted influenza virus vaccine for humans. Rev Med Virol. 1999 October-December; 9(4):237-44. Review. PubMed PMID: 10578119; Gendon IuZ. [Live cold-adapted influenza vaccine: state-of-the-art]. Vopr Virusol. 2011 January-February; 56(1):4-17. Review. Russian. PubMed PMID: 21427948; Aleksandrova G I, Gushchina M I, Klimov A I, Iotov V V. [Genetic basis for construction of the life influenza type A vaccine using temperature-sensitive mutants]. Mol Gen Mikrobiol Virusol. 1990 March; (3):3-8. Review. Russian. PubMed PMID: 2194119; Kendal A P. Cold-adapted live attenuated influenza vaccines developed in Russia: can they contribute to meeting the needs for influenza control in other countries? Eur J Epidemiol. 1997 July; 13(5):591-609. Review. PubMed PMID: 9258574).
Unlike licensed live influenza vaccines (LIVE allantoic INFLUENZA VACCINE ULTRAVAC® (RF) or Flumist® (USA)), known influenza vectors NS1-124 and constructions close to them did not possess the phenotypic temperature-sensitivity marker (ts phenotype) and had levels of reproduction in mouse lungs, close to the level of the wild-type virus with the full-length NS1 protein.
In 50-60th years of the 20th century, attempts were made to use influenza viruses as an oncolytic agent, which were based on the physician's observations of individual cases of cancer remission after recovering from influenza infection (Lindenmann J, Klein P A. Viral oncolysis: increased immunogenicity of host cellantigen associated with influenza virus. J Exp Med. 1967 Jul. 1; 126(1):93-108).
Since the development of genetic engineering techniques for influenza virus in the late 90s, this created a possibility of producing oncolytic influenza vectors with a modified NS1 protein. It was shown that truncation of the NS1 protein could lead to an enhancement in the oncolytic effect when introducing a recombinant virus into a tumor, due to stimulation of the innate immune system to which the NS1 protein is an antagonist (Sturlan S, Stremitzer S, Bauman S, Sachet M, Wolschek M, Ruthsatz T, Egorov A, Bergmann M. Endogenous expression of proteases in colon cancer cells facilitate influenza A viruses mediated oncolysis. Cancer Biol Ther. 2010 Sep. 15; 10(6):592-9; Ogbomo H, Michaelis M, Geiler J, van Rikxoort M, Muster T, Egorov A, Doerr H W, Cinatl J Jr. Tumor cells infected with oncolytic influenza A virus prime natural killer cells for lysis of resistant tumor cells. Med Microbiol Immunol. 2010 May; 199(2):93-101. doi: 10.1007/s00430-009-0139-0. Epub 2009 Dec. 15. PubMed PMID: 20012989; Efferson C L, Tsuda N, Kawano K, Nistal-Villán E, Sellappan S, Yu D, Murray J L, García-Sastre A, Ioannides C G. Prostate tumor cells infected with a recombinant influenza virus expressing a truncated NS1 protein activate cytolytic CD8+ cells to recognize noninfected tumor cells. J Virol. 2006 January; 80(1):383-94).
Moreover, the possibility of genetic engineering manipulations with the length of the influenza virus NS1 protein allowed the development of vectors whose effectiveness enhanced by the presence of the expression of an immunopotentiating agent, for example interleukin-15 (van Rikxoort M, Michaelis M, Wolschek M, Muster T, Egorov A, Seipelt J, Doerr H W, Cinatl J Jr. Oncolytic effects of a novel influenza A virus expressing interleukin-15 from the NS reading frame. PLoS One. 2012; 7(5):e36506).
These works unfortunately used influenza viruses capable of limited reproduction in some cell cultures that do not possess a necessary genetic stability of the transgene for large-scale production in chicken embryos, which are a substrate optimal for the production of influenza vaccine preparations.
Thus, there remains a need for new effective viral vectors, in particular attenuated influenza vectors, that are characterized by the lack of active reproduction of the virus in animal organisms and have temperature-sensitivity phenotype, and that can be used for the prevention and/or treatment of infectious diseases, as well as for the treatment of oncological diseases.